DESCRIPTION(From applicant's abstract): This is a renewal application of our grant DA00564-28 which was committed to study the neurochemical mechanisms of narcotic tolerance since its inception. Though under intensive investigation, the molecular mechanism of opioid tolerance and dependence has yet to be determined. A general concept of homeostasis has been the focus of much discussion, i.e. drug exposure disrupts the balance of the system, thus tolerance and dependence are cellular adaptations to regain the homeostasis. There are many theories and experimental data in support of different hypotheses such as transcriptional regulation leading to macromolecule synthesis or the alteration in the neurotransmission. In this proposal, we test the hypothesis that opioid tolerance stems from the decrease in the receptor signaling during chronic treatment, or receptor desensitization. The uncoupling of the receptor from the effectors might not be the case in the long-term effects of the drug, but it is the most efficient manner to regain the system's homeostasis. During the past funding period, by generating the mu opioid receptor knockout animals, we conclusively demonstrated that the morphine in vivo activities are absolutely dependent on the concentration of the mu opioid receptor. In parallel studies, we identified the agonist-induced phosphorylation sites of the mu opioid receptor in the in vitro cell lines models. Agonists, such as morphine, induced the phosphorylation of Thr370 and Ser375 residues within the carboxyl domain of the mu-receptor. A basal phosphorylation at the Ser363 was also observed. Now, we are in the position to address the functional roles of the phosphorylation of these critical Ser/Thr residues. We will investigate the roles of these residues in the rapid and slow desensitization of the mu-opioid receptor. We will investigate the existence of these residues in the agonist-induced internalization of the receptor. We will investigate the existence of possible cell line specificity in the phosphorylation of these residues by using various clonal cell lines of primary in vitro neuronal cultures transiently transfected with the adenovirus containing the wild type or mutant receptors. We will determine the identity of the kinases that might be involved in the phosphorylation of these residues. Finally, we will generate mutant receptor in vivo by the gene targeting approach to address the question of the receptor phosphorylation on opioid tolerance development in vivo. Hence, we anticipate that, by carrying out both the in vitro and in vivo studies, we will be able to demonstrate opioid tolerance is a consequence of receptor signaling decrease, or receptor desensitization.